Engineering an artificial catch bond using mechanical anisotropy

Catch bonds are a rare class of protein-protein interactions where the bond lifetime increases under an external pulling force. Here, we report how modification of anchor geometry generates catch bonding behavior for the mechanostable Dockerin G:Cohesin E (DocG:CohE) adhesion complex found on human gut bacteria. Using AFM single-molecule force spectroscopy in combination with bioorthogonal click chemistry, we mechanically dissociate the complex using five precisely controlled anchor geometries. When tension is applied between residue #13 on CohE and the N-terminus of DocG, the complex behaves as a two-state catch bond, while in all other tested pulling geometries, including the native configuration, it behaves as a slip bond. We use a kinetic Monte Carlo model with experimentally derived parameters to simulate rupture force and lifetime distributions, achieving strong agreement with experiments. Single-molecule FRET measurements further demonstrate that the complex does not exhibit dual binding mode behavior at equilibrium but unbinds along multiple pathways under force. Together, these results show how mechanical anisotropy and anchor point selection can be used to engineer artificial catch bonds.

), which is in contrast to the native anchor geometry.

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Figure S2 SDS-PAGE showing conjugation of Fgβ to Coh F13AzF mutant.The Coh F13AzF mutant (Lane 1) was conjugated with Fgβ peptide.The reaction product (Lane 2) was subsequently purified using a size-exclusion column to remove the excess Fgβ peptide (Lane 3), and a Strep-trap column to remove unconjugated Coh F13AzF (Lane 4).

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Figure S3 Rupture force histograms measured at different anchor geometries and different pulling speeds.The histograms were fitted with one (anchor geometry NN-B, pulling from CohE N-terminus and DocG N-terminus) or two (other anchor geometries) Gaussian peaks.

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Figure S4 Linear regression between the percentage of high force population and the logarithm of pulling speed.Only anchor geometries exhibiting bimodal rupture force distribution are shown: native anchor geometry (a), and non-native anchor geometries NN-A (b), NN-C (c), and NN-D (d).For each anchor geometry, the prevalence of the high force population measured at different pulling speeds are plotted against the logarithm of the pulling speed and fitted linearly.The Pearson's correlation coefficients r and the p-values of Analysis of Variance (ANOVA) test are shown for each anchor geometry and summarized in TableS2.Only anchor geometry NN-C (panel c) has a p-value<0.05,meaning that the slope of linear fitting is significantly larger than zero.

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Figure S5 Native anchor geometry measurement of DocG:CohE (F13-Fgβ).a: AFM-SMFS experimental setup.The residue F13 of CohE in CohE-FLN-ELP-ybbr construct was replaced by azido-phenylalanine and conjugated with Fgβ peptide, and the construct was immobilized on the AFM tip.ybbr-ELP-FLN-DocG construct was immobilized on the glass surface.The rupture force of DocE:CohG (F13-Fgβ) complex was measured at the native anchor geometry and at different pulling speeds.b: The force-loading rate plot of DocG:CohE (F13-Fgβ).The average rupture forces of the high force and low force pathways measured at four different pulling speeds were linearly fitted against loading rate to extract energy landscape parameters.The n number represents the total number of single protein complexes measured.c: Rupture force histograms of DocG:CohE (F13-Fgβ) at different pulling speeds.The n numbers represent the number of single protein complexes measured at each pulling speed.d: Fraction of high force pathway at different pulling speeds.The n number represents the total number of single protein complexes measured.

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Figure S6 Overlay of force-loading rate plots of experimental and simulation results.The Monte Carlo simulation results for the force-loading rate relationship at the native (a) and catch (b) anchor geometries are overlayed with the corresponding experimental results.The simulation results are shown in black dots and the experimental data are shown in blue (native anchor geometry) and brown (catch anchor geometry) squares.The Bell-Evans fitting results are shown in solid line (high-force population) and dashed line (low-force population).

Figure S7
Figure S7 Monte Carlo simulation rupture force histograms.a: Rupture force histograms of DocG:CohE at native anchor geometry (upper panels) and catch anchor geometry (corresponding to the non-native anchor geometry NN-C, lower panels).Each histogram was fitted in two Gaussian peaks.b: The prevalence of high rupture force population at different pulling speeds in Monte Carlo simulation.At the catch anchor geometry, the prevalence of high force population increases with increasing pulling speed (ANOVA test of linear regression p < 0.05, see TableS2), which